\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[ngerman,greek,polish,english]{babel}



\usepackage{float}








\begin{document}

\title{Glucose oxidase virus-based nanoreactors for smart breast cancer therapy}



\author[1]{Pedro Gama}%
\author[2]{Patrcica Juarez}%
\author[1]{Ana Rodríguez-Hernández}%
\author[1]{Rafael Vazquez-Duhalt}%
\affil[1]{Universidad Nacional Autónoma de México Centro de Nanociencias y Nanotecnología}%
\affil[2]{Centro de Investigacion Cientifica y de Educacion Superior de Ensenada}%


\vspace{-1em}



  
  \date{May 20, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Background Breast cancer is the most common malignant tumor disease and
the leading cause of female mortality. The evolution of nanomaterials
science opens the opportunity to improve traditional cancer therapies,
enhancing therapy efficiency and reducing side effects. Methods and
major results Herein, protein cages conceived as enzymatic nanoreactors
were designed and produced by using virus-like nanoparticles (VLPs) from
Brome Mosaic Virus (BMV) and containing the catalytic activity of
glucose oxidase enzyme (GOx). The GOx enzyme was encapsulated into the
BMV capsid (VLP-GOx), and the resulting enzymatic nanoreactors were
coated with human serum albumin (VLP-GOx@HSA) for breast tumor cell
targeting. The effect of the synthesized GOx nanoreactors on breast
tumor cell lines was studied in vitro. Both nanoreactor preparations
VLP-GOx and VLP-GOx@HSA showed to be highly cytotoxic for breast tumor
cell cultures. Cytotoxicity for human embryonic kidney cells was also
found. The monitoring of nanoreactors treatement on triple negative
breast cancer cells showed an evident production of oxygen by the
catalase antioxidant enzyme induced by the high production of hydrogen
peroxide from GOx activity. Conclusions and implications The
nanoreactors containing GOx activity are fully suitable for cytotoxicity
generation in tumor cells. The HSA functionalization of the VLP-GOx
nanoreactors could result in a prevailing strategy to improve selective
cancer targeting. The GOx containing enzymatic nanoreactors seems to be
an interesting alternative to improve the current cancer therapy. In
vivo studies are on going to reinforce the effectiveness of this
treatment strategy.%
\end{abstract}%



\sloppy


\textbf{Glucose oxidase virus-based nanoreactors for smart breast cancer
therapy}

Pedro Gama \textsuperscript{1,2}, Patricia Ju\selectlanguage{ngerman}árez \textsuperscript{2},
Ana G. Rodríguez-Hernández, and Rafael
Vazquez-Duhalt\textsuperscript{1}*.

\textsuperscript{1} Centro de Nanociencias y Nanotecnología, Universidad
Nacional Autónoma de México, Km 107 Carretera Tijuana-Ensenada,
Ensenada, Baja CA 22860, Mexico.

\textsuperscript{2} Centro de Investigación Científica y de Educación
Superior de Ensenada, Baja California.

\textbf{* Corresponding author}

Prof. Rafael Vazquez-Duhalt

Centro de Nanociencias y Nanotecnología UNAM

Km 107 carretera Tijuana-Ensenada

Ensenada. Baja California

22760 México

Email: \emph{rvd@ens.cnyn.unam.mx}

\textbf{Keywords:} Breast cancer, Brome mosaic virus, Enzymatic
nanoreactors, Glucose oxidase, Protein cage, Virus-like particle.

\textbf{Abbreviations}

BMV, Brome Mosaic Virus

CP, coat protein

DLS, dynamic light scattering

GOx, glucose oxidase

HSA human serum albumin

VLP, virus-like nanoparticle



\textbf{Abstract}

Background

Breast cancer is the most common malignant tumor disease and the leading
cause of female mortality. The evolution of nanomaterials science opens
the opportunity to improve traditional cancer therapies, enhancing
therapy efficiency and reducing side effects.

Methods and major results

Herein, protein cages conceived as enzymatic nanoreactors were designed
and produced by using virus-like nanoparticles (VLPs) from Brome Mosaic
Virus (BMV) and containing the catalytic activity of glucose oxidase
enzyme (GOx). The GOx enzyme was encapsulated into the BMV capsid
(VLP-GOx), and the resulting enzymatic nanoreactors were coated with
human serum albumin (VLP-GOx@HSA) for breast tumor cell targeting. The
effect of the synthesized GOx nanoreactors on breast tumor cell lines
was studied \emph{in vitro} . Both nanoreactor preparations VLP-GOx and
VLP-GOx@HSA showed to be highly cytotoxic for breast tumor cell
cultures. Cytotoxicity for human embryonic kidney cells was also found.
The monitoring of nanoreactor treatment on triple-negative breast cancer
cells showed an evident production of oxygen by the catalase antioxidant
enzyme induced by the high production of hydrogen peroxide from GOx
activity.

Conclusions and implications

The nanoreactors containing GOx activity are fully suitable for
cytotoxicity generation in tumor cells. The HSA functionalization of the
VLP-GOx nanoreactors could result in a prevailing strategy to improve
selective cancer targeting. The GOx containing enzymatic nanoreactors
seems to be an interesting alternative to improve the current cancer
therapy. \emph{In vivo} studies are ongoing to reinforce the
effectiveness of this treatment strategy.



\textbf{1. Introduction}

Breast cancer is the most common malignant tumor worldwide and the
leading cause of female mortality.\textsuperscript{{[}1,2{]}} More than
half a million deaths caused by breast cancer were recorded in 2020 by
the Global Cancer Center at WHO.\textsuperscript{{[}3{]}} This
histological pathology implies the appearance of adenocarcinoma in
mammary glands.\textsuperscript{{[}4{]}} Current therapy for breast
cancer consists of invasive tumor surgical resection and systemic
chemotherapy treatment.\textsuperscript{{[}5{]}} However, non-specific
chemo-pharmacologic treatments induce several drastic side
effects.\textsuperscript{{[}6,7{]}} Today, nanomedicine is focusing on
designing novel and smart therapies to increase the efficiency of breast
cancer treatment.\textsuperscript{{[}8-10{]}}

Glucose oxidase (GOx) enzyme activity has been recently proposed for
cancer nanomedicine applications. GOx, an intracellular enzyme
from\emph{Aspergillus niger} is widely used in glucose determination,
fermentation industry, biosensors fabrication, and as a potential
antibiotic.\textsuperscript{{[}11,12{]}} This enzyme catalyzes glucose
oxidation to produce hydrogen peroxide and gluconolactone. In a tumor
environment, the GOx activity reduces, both available glucose and
molecular oxygen, essential compounds for cell metabolism. In addition,
GOx produces oxidative stress generating hydrogen peroxide, killing
tumor cells.\textsuperscript{{[}13{]}} Different designs of GOx enzyme
systems have been recently developed for cancer therapy, including
several mono and multimodal vesicles, polymer dots, and magnetic
nanoparticles.\textsuperscript{{[}14-17{]}}

Nanoparticle specific-targeting is an emerging field to deliver
cytotoxic activity to tumor cells selectively. The specific
anticancer-drug delivery increases the treatment effectiveness and
reduces the drastic side effects.\textsuperscript{{[}18{]}} A diversity
of nanosystems, such as polymer, protein, metallic, organic, and
inorganic nanoparticles, has been proposed for biomedical
applications.\textsuperscript{{[}19{]}} Protein cages based on viral
capsids, or virus-like particles (VLPs), are interesting nanosystems
that have been proposed as nanocarriers for delivering anticancer
therapies to specific tumor cells.\textsuperscript{{[}20,21{]}} VLPs are
widely used in vaccine technology and have recently recently been
proposed as nanoplatforms as carriers of drugs for different biomedical
therapies.\textsuperscript{{[}22-25{]}} VLPs can be derived from native
viruses or obtained by recombinant technology. After removing the
genetic material, the purified monomeric coat protein is self-assembled
under certain conditions forming hollow nanoparticles. Self-assembly
property is used to confine or encapsulate several cargo molecules
producing well-defined symmetry and homogenous size nanoparticles. The
VLPs are highly stable in carrying and delivering cargo molecules, are
biocompatible and biodegradable, and show low
toxicity.\textsuperscript{{[}26,27{]}} The surface of VLPs can be
functionalized with a diversity of ligands to be specifically targeted
to cells and tissues \textsuperscript{{[}28,29{]}} making smarter and
more efficient therapies. In addition, the suspension of VLPs is highly
stable in biological fluids.

VLPs can contain active enzymes, and the arrangement is called an
enzymatic nanoreactor.\textsuperscript{{[}22{]}} Improved catalytic
properties have been reported for virus-based enzymatic
nanoreactors.\textsuperscript{{[}30-32{]}} VLP-based enzymatic
nanoreactors containing cytochrome P450 activity have been proposed for
prodrug activation in breast cancer
therapy.\textsuperscript{{[}33-35{]}} The protein cage structure
protects the catalytic molecule from protease degradation and decreases
enzyme recognition by the immune system conferring a better catalytic
performance.\textsuperscript{{[}36-38{]}} Moreover, the intrinsic porous
structure of the VLP-based nanoreactors can allow the substrates and
products to flow through the system.\textsuperscript{{[}39-41{]}}

This work encapsulated glucose oxidase in VLPs from Brome Mosaic Virus
(BMV). The catalytic properties of the enzymatic nanoreactors were
analyzed, and their effect on tumor cell lines was determined.



\textbf{2. Materials and Methods}

2.1 Materials

Type VII glucose oxidase from \emph{Aspergillus niger} , human serum
albumin (HSA, 96\%), dextrose (99.5\%), hydrogen peroxide (30 wt\%),
guaiacol (99\%), L-glutathione reduced (98\%), and all other general
reagents, precursors and solvents were purchased from Sigma-Aldrich (St.
Louis, MO). Salts used for buffer solutions were purchased at Fermont
(Monterrey, MX). TEM analysis was performed using copper grids
(400-mesh) coated with formvar/carbon support film (Ted-Pella, USA). All
chemicals were of the highest grade commercially available. Cancer cell
lines were acquired at ATTC (USA).

2.2 BMV virus production

The VLP particles were produced and purified from the native virus as
previously reported.\textsuperscript{{[}42{]}} The BMV native virus was
amplified through plant infection to produce VLPs. First, barley plants
(\emph{Hordeum vulgare} ) were grown under controlled conditions. Then,
young barley leaves were inoculated with the BMV virus. Once the leaves
showed the infection pattern, they were harvested and stored at -20°C to
be later liquefied in a blender (Osterizer) with virus extraction buffer
(0.5 M sodium acetate, 80 mM magnesium acetate, pH 4.5). The resulting
macerated material was filtered through cheesecloth, then the
flowthrough was mixed with chloroform 1:1 v/v. The mixture was
centrifuged at 10,000 rpm, 4°C, for 20 min. Since two phases formed in
the centrifuge bottle; the upper phase (aqueous phase) was collected and
stirred at 4°C overnight. Next, the aqueous extract containing the BMV
virions was ultracentrifuged on a 5 mL sucrose cushion in a 32 mL
centrifuge tube at 32,000 rpm, 4°C for 2 h. After centrifugation, the
supernatant was discarded and a pellet containing the BMV virions was
resuspended in a virus suspension buffer (50 mM sodium acetate, 8 mM
magnesium acetate, pH 4.5). Then, the BMV virion suspension was poured
into a previously prepared centrifuge tube with a sucrose density
gradient (10-40\%) and centrifuged at 30,000 rpm, 4°C for 2 h. After
centrifugation, the tube was examined under white light to confirm the
presence of the blue phase containing the viruses. The blue phase was
then recovered and diluted 1:4 v/v in a virus suspension buffer. The
dilute blue phase was centrifuged at 32,000 rpm, 4°C for 3 h to
concentrate the virions in a pellet. After centrifugation, the
supernatant was discarded and the pellet containing the virions was
resuspended in virus suspension buffer and stored at -70°C.

2.3 Capsid protein (CP) purification

The native BMV virions were disassembled to obtain the CP protein needed
to synthesize the VLPs. First, the BMV virions were dialyzed against the
disassembly buffer (0.5 M CaCl\textsubscript{2}, 50 mM Tris-HCl, pH 7.4)
for 5 h. Then, the resulting disassembled CP was centrifuged at 50,000
rpm, 4°C for 4 h. After centrifugation, the supernatant containing the
CP was collected in 0.5 mL fractions. The CP purity of the fractions was
estimated using the A\textsubscript{280}/A\textsubscript{260} nm
quotient. The best fractions (A\textsubscript{280}/A\textsubscript{260}\selectlanguage{english}[?]
1.6) were combined and dialyzed against the protein buffer (1 M NaCl, 20
mM Tris-HCl, pH 7.2) for 2 h. Finally, the dialyzed CP was recovered and
then quantified by absorbance using the CP extinction coefficient.

2.4 VLP-GOx nanoreactors synthesis

The VLP-GOx nanoreactors were synthesized by combining the disassembled
BMV capsid and free-GOx enzyme and using the auto-assembly method.
First, the disassembled CP protein was mixed with the free-GOx enzyme
(in phosphate buffer pH 7.0) at different molar ratios (1:2, 1:3, 1:6,
and 1:23 GOx:CP). Then, the GOx:CP mixtures were dialyzed in two steps.
First, initial buffer (50 mM NaCl, 10 mM KCl, 5 mM
MgCl\textsubscript{2}, 50 mM Tris, pH 7.2); and second, auto-assembly
buffer (50 mM sodium acetate, 750 mM NaCl, 8 mM magnesium acetate, pH
5.1), 4 h, 4degC. After dialysis, the samples were centrifuged at 40,000
rpm, 4degC for 2 h to obtain the nanoreactors in a pellet. After
centrifugation, the supernatant was removed and the pellet containing
the VLP-GOx nanoreactors was resuspended in the auto-assembly buffer.
The resulting VLP-GOx nanoreactor suspensions were filtered using a 0.2
\selectlanguage{greek}µ\selectlanguage{english}m filter for further high-performance liquid chromatography (HPLC)
purification. Next, the nanoreactors suspension was injected in a size
exclusion column (TSKgel Column, Super SW2000, Supelco-Tosoh Bioscience,
Bellefont, PA) for HPLC (Agilent 1100 series) purification of the
VLP-GOx nanoreactors. The VLP-GOx nanoreactor fractions were collected
and then ultrafiltered through a 30,000 Da membrane (Merck Amicon
Ultra-15 Centrifugal Filter Units) to adjust the work concentration.
Finally, the resulting VLP-GOx nanoreactors were estimated by Dynamic
Light Scattering (DLS) with a Zetasizer Nanoseries device (Nano-ZS,
Malvern Instruments). The total amount of protein in all preparations
was estimated via the Bio-Rad Bradford Protein Assay (Hercules, CA).

2.5 VLP-GOx surface functionalization

The VLP-GOx nanoreactors were prepared by functionalizing their surface
to improve their affinity to breast cancer cells. Hence, the outer
surface of the VLP-GOx nanocages was covered with human serum albumin
(HSA) protein. First, the HSA was activated with reduced glutathione as
reported by Chauhan, et al, 2020. Second, the VLP-GOx nanoreactors
(synthesized at 1:3 molar ratio) and reduced HSA were mixed at 1:8
VLP:HSA w/w ratio and stirred overnight at 4\selectlanguage{ngerman}°C for the coating reaction.
Next, the HSA functionalized nanoreactors were centrifuged at 40,000
rpm, 4°C for 2 h. After centrifugation, the supernatant was discarded
and a pellet containing the HSA functionalized VLP-GOx nanoreactors
(VLP-GOx@HSA) was resuspended in the assembly buffer. Finally, the
VLP-GOx@HSA suspension was purified by HPLC to get rid of free-HAS and
aberrant capsids.

2.6 GOx Nanoreactors Characterization

The nanoreactor catalytic activity was characterized by a
peroxidase-coupled reaction followed by a colorimetric assay. The last
product in this coupled reaction is tetraguaiacol, which absorbs at 470
nm. The increased absorbance is directly related to the glucose
transformation rate. A Michaelis-Menten regression was performed by
preparing reactions at different glucose concentrations (0, 25, 50, 100,
and 150 mM) to describe the catalytic parameters of the VLP-GOx
nanoreactors.

The encapsulation of the GOx enzyme in VLP nanoparticles and the HSA
coating was confirmed by SDP-PAGE. Finally, the size and morphology of
the nanoreactor were studied with transmission electron microscopy (TEM,
JEOL-2010, JEOL) operated at 100 kV.

2.7 Cytotoxic effects of nanoreactors

Breast tumor cell cytotoxic effects of GOx nanoreactors were studied by
means of an \emph{in vitro} assay. Two cell lines were exposed to
different nanoreactor amounts. First, human embryonic kidney (293T) as a
human healthy cell model; second 4T1 (murine breast cancer tumor). The
nanoreactor-exposed cells were analyzed by an MTT colorimetric assay
(TOX-1, Sigma-Aldrich, St. Louis, MO) to estimate the cytotoxic effect.
Firstly, the cells were cultured in 96 well-plates; for 293T, 10,000
cells/well; for 4T1, 4,000 cells/well at 37°C and 5\%
CO\textsubscript{2}. After 24 h, the cells were treated with different
nanoreactor amounts. Finally, 48 h later starting the cultures, the MTT
reagent (methyl-134-thiazolyl-tetrazolium) was added to the cells, and
the absorbance (570 nm) showed in the well-plate was measured with a
Multiskan GO spectrophotometer (Thermo Scientific). The resulting
absorbance from negative control (cell media) was established as 100\%
of cell survival. Then, experimental groups were compared to estimate a
survival rate.



\textbf{2.8 Fluorescence microscopy}

Cell culture of 5x10\textsuperscript{5} of MDA-MB231/eGPF (Cell Biolabs
Inc, Ref. AKR-201) was seeded onto cell culture-treated Petri dishes (3
cm of diameter) and incubated in DMEM cultute medium supplement with
10\% foetal bovine serum (Biowest, France) and 1\% of
antibiotic/antimycotic (GIBGO) overnight at 37°C under 5\%
CO\textsubscript{2} atmosphere. Afterward, cells were washed with
phosphate buffer saline (PBS) 1X. Then, 100 \selectlanguage{greek}µ\selectlanguage{english}g of VLP-GOx resuspended in
DMEM were added to the cell culture and incubated. Time-lapse images of
cells were obtained by LS720 Microscope Lumascope (Etaluma Inc., San
Diego, CA) every 2 minutes for 4 hours. Phase contrast and green
fluorescent (green filter 473-491/502-561 excitation/emission) images
were obtained.

\textbf{2.9 Statistical analysis}

Differences between data were tested using a two-way repeated measures
(RM) ANOVA with Sidak or Tukey post hoc tests as appropriate.
Statistically significant differences were accepted as P-values of
\textless{} 0.001 and Excel statistics (Microsoft, Redmond, WA) was used
for all analysis.



\textbf{3. Results}

3.1 Enzymatic nanoreactor synthesis and functionalization

The complementary electrostatic charge between the negatively charged
GOx enzyme surface and the positively charged inner face of the BMV
capsid drive the enzyme encapsulation (Fig. 1a). The isoelectric point
of GOx was determined by electrophoretic light scattering using the Z
potential at different pH and showed to be 4.6 (SI 1). The self-assembly
process was modulated by ionic strength (0.75 M) at pH 5.1. The GOx
enzyme was successfully encapsulated inside VLPs from BMV by the
self-assembly synthesis method (Fig. 1b).

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/614980/articles/641602-glucose-oxidase-virus-based-nanoreactors-for-smart-breast-cancer-therapy}

\textbf{Figure 1. a)} Surface electric charge distribution on the GOx
enzyme and BMV capsomer. The GOx surface mostly exhibits negative
electric charges (red color), and the inner surface of BMV capsomers
shows a more positive (blue color) electric charge distribution. The
difference in electric charge distribution between GOx and the inner BMV
surface results in electric charge complementarity. \emph{In
silico}results by Maestro Schrodinger\selectlanguage{ngerman}®. b) Schematic representation of
VLP-GOx nanoreactor self-assembly encapsulation and HSA
functionalization.

To optimize the enzyme encapsulation, four different enzyme-coat protein
molar ratios (GOx:CP) were assayed (Table 1). Nanoreactors containing
enzymatic activity (VLP-GOx) were obtained in all mixtures tested. All
nanoreactor preparations were purified by size exclusion chromatography
(SEC) with an HPLC (Fig. 2). The purified preparation showing the
highest enzymatic activity (1:3 molar ratio) was then functionalized
with human serum albumin (HSA) (Fig. 1b). HSA shows binding affinity for
specific receptors on the surface of different cells in diseased organs
that permits the active targeting and the specific recognition of the
albumin-based formulations. \emph{In vitro} and \emph{in
vivo}experiments HSA conjugated preparations showed higher cellular
uptake efficiency, longer half-life, higher cytotoxicity, and
accumulation in tumors to a much greater extent than the free drug
preparations.\textsuperscript{{[}43, 44{]}} The functionalization was
carried out through previously reduced cysteine groups of HSA protein,
producing di-thiol cross-linking with the cysteine (Cys108) of CP-BMV
(SI 2).

\textbf{Table 1.} Hydrodynamic diameter determined by Dynamic Light
Scattering (DLS) of proteins and catalytic contants of enzymatic
nanoreactors assembled at different GOx:CP molar ratios.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Preparation}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Preparation}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Hydrodymanic diameter} \textbf{(nm)}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{V\textsubscript{max}} \textbf{(U/g protein)}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{K\textsubscript{M}} \textbf{(mM)}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Free GOx\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Free GOx\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
12.2 \selectlanguage{ngerman}±4.1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
237,985\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
27.8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Coat protein (CP)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Coat protein (CP)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
6.8 \selectlanguage{ngerman}±1.2\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
--\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Native BMV\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Native BMV\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
27.6 \selectlanguage{ngerman}±6.7\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
--\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
--\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{GOx:CP molar ratio}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{VLP-GOx nanoreactors}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{VLP-GOx nanoreactors}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{VLP-GOx nanoreactors}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{VLP-GOx nanoreactors}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1:2\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1:2\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
25.8 \selectlanguage{ngerman}±8.5\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
507\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
47.7\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1:3\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1:3\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
28.8 \selectlanguage{ngerman}±7.9\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1,208\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
34.6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1:6\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1:6\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
33.4 \selectlanguage{ngerman}±14.2\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
872\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
40.0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1:23\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1:23\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
26.1 \selectlanguage{ngerman}±8.9\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
137\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
49.0\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1:3 VLP-GOx@HSA\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1:3 VLP-GOx@HSA\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
29.7 \selectlanguage{ngerman}±8.9\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
106\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
70.4\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\emph{}

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/614980/articles/641602-glucose-oxidase-virus-based-nanoreactors-for-smart-breast-cancer-therapy}

\textbf{Figure 2}. Purification of VLP-GOx nanoreactors by SEC HPLC
chromatography using differences in retention time \textbf{A)}Overlapped
chromatograms show the retention time for native BMV=5.7 min, free
GOx=7.5 min and disassembled CP=8.4 min. \textbf{B)} VLP-GOx
nanoreactors retention time=5.7 min, similar to native virions of BMV.
The VLP-GOx nanoreactors were purified by collecting the fractions from
5.5 to 6.5 min.

3.2 Nanoreactors characterization

The nanoreactor's hydrodynamic size was estimated by Dynamic Light
Scattering (DLS). No significant changes in hydrodynamic diameters were
found. All the assayed ratios produced VLP-GOx particles with a similar
hydrodynamic diameter to the native virus ([?]28 nm). The unfunctionalized
VLP-GOx showed a hydrodynamic diameter of 28.8 \selectlanguage{ngerman}±7.9 nm, while the
functionalized VLP-GOx@HSA showed a value of 29.7 ±13.3 nm (Table 1).

The structure of the VLP-GOx nanoreactors was also characterized by
transmission electron microscopy (TEM) (Fig. 3). The obtained
micrographs of the VLP-GOx nanoreactor show a quasi-spherical shape with
similar symmetry to the native BMV virus (28 nm). In addition,
functionalized VLP-GOx@HSA nanoreactors show an ornamented outer surface
displaying HSA protein molecules on the nanoreactor surface.

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/614980/articles/641602-glucose-oxidase-virus-based-nanoreactors-for-smart-breast-cancer-therapy}

\textbf{Figure 3}. Transmission electron microscopy (TEM) of
nanoreactors. A) Quasi-spherical unmodified VLP-GOx nanoreactors, and B)
VLP-GOx@HSA functionalized nanoreactors.

3.3 Catalytic characterization

The protein proportions in the nanoreactors were estimated by
densitometry on an SDS-PAGE gel. According to the band's intensity, the
GOx enzyme in the unfunctionalized VLP-GOx corresponds to 3.58\% of the
total protein, while in the HSA functionalized preparations
(VLP-GOx@HSA) the GOx represented only 0.82\%, coat protein (CP) 22.8\%
and the cover shell of HSA 76.38\% of the total protein in the
nanoreactor.

The catalytic constants for GOx activity of nanoreactors were estimated
using a Michaelis-Menten nonlinear regression. The assembly from 1:3
GOx:CP molar ratio showed the highest specific activity (1,208 U/g) and
a Michaelis constant (K\textsubscript{M}) of 34.6 mM (Table 1). Thus,
this molar ratio was used for further studies and for HSA
functionalization. Considering the GOx content in the nanoreactor
preparations, the V\textsubscript{max} of VLP-GOx, in enzyme activity
basis, is estimated to be 33,743 U/g of GOx, representing 14\% of the
transformation rate of free enzyme. On the other hand, the
V\textsubscript{max} of functionalized VLP-GOx@HSA en enzyme basis is
12,990 U/g of GOx, 5.5\% of the value of the free enzyme. Using the
amount of enzyme contained in the nanoreactors, the transformation rate
constant (k\textsubscript{cat}) could be obtained. The free enzyme
showed a k\textsubscript{cat} value of 634 s\textsuperscript{-1}, while
the preparations VLP-GOx and VLP-GOx@HSA showed values of 90
s\textsuperscript{-1} and 34 s\textsuperscript{-1}, respectively. In
addition, it is possible to estimate the number of GOx molecules (active
dimer of 160,000 Da) per capsid or nanoreactor (180 monomers of 20,385
Da). The number of GOx molecules inside each VLP was estimated as an
average of 0.85 molecules of GOx per VPL nanoreactor.

3.4 Antitumoral effect

The antitumoral effect of nanoreactors was assayed in 4T1 breast tumor
cells, a breast cancer cell line derived from the mammary gland tissue
of a mouse. In addition, the cytotoxic effect of nanoreactors was also
assayed on cultures of 293T healthy cell line from the human embryonic
kidney. Cytotoxicity was directly estimated as the cell viability as a
metabolic activity using the MTT colorimetric assay.

Control experiments were carried out with VLP deprovided of GOx (Fig.
4). Interestingly, high concentrations of empty VLPs and VLPs@HSA
(\textgreater{}25 \selectlanguage{greek}µ\selectlanguage{english}g/mL) significantly reduced the cell viability of 4T1
cancer cells. In contrast, VLPs treatment of 293T epithelial cells did
not reduce cell viability at the highest concentration tested of 100
\selectlanguage{greek}µ\selectlanguage{english}g/mL, except for unfunctionalized VLPs.

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/614980/articles/641602-glucose-oxidase-virus-based-nanoreactors-for-smart-breast-cancer-therapy}

\textbf{Figure 4} . Cytotoxicity activity of empty unmodified and
modified VLPs on 4T1 breast tumor mouse cell line model, and 293T
healthy embryonic kidney human cells. Two-way RM ANOVA
(p\textless{}0.001). *** Significative difference between the two
preparations at the same concentration. ++ Significative difference with
the control.

The same cytotoxicity experiments were performed with GOx-containing
nanoreactors (Figs. 5 and 6). The results were expressed based on both
GOx content and nanoreactor GOx activity. These two forms to express the
treatment doses are due because the protein content of the two
preparations is significantly different, and if the GOx content is
similar in capsid base the enzymatic activity changes when the
nanoreactor is covered with HAS (Table 1). On the 4T1 breast tumor
cells, the LD\textsubscript{50} values were 0.04 \selectlanguage{greek}µ\selectlanguage{english}g GOx/mL for VLP-GOx
and around 0.1 \selectlanguage{greek}µ\selectlanguage{english}g GOx/mL for VLP-GOx@HSA, and no metabolic activity was
detected at 1 \selectlanguage{greek}µ\selectlanguage{english}g GOx/mL for both preparations (Fig. 5a). The 4T1 tumor
cells showed an LD\textsubscript{50} at around 1 mU of GOx activity per
mL of cell culture, and no metabolic activity at around 10 mU/mL in both
preparations (Fig. 5b).

\textbf{Hosted file}

\verb`image5.emf`  available at \url{https://authorea.com/users/614980/articles/641602-glucose-oxidase-virus-based-nanoreactors-for-smart-breast-cancer-therapy}

\textbf{Figure 5} . Cytotoxicity of unfunctionalized VLP-GOx and
functionalized VLP-GOx@HSA nanoreactors on 4T1 breast tumor mouse cell
line.

\textbf{Hosted file}

\verb`image6.emf`  available at \url{https://authorea.com/users/614980/articles/641602-glucose-oxidase-virus-based-nanoreactors-for-smart-breast-cancer-therapy}

\textbf{Figure 6} . Cytotoxicity of unfunctionalized VLP-GOx and
functionalized VLP-GOx@HSA nanoreactors on 293T kidney cell line.

The cytotoxicity was also assayed in 293T healthy embryonic kidney human
cells (Fig. 6). The results were expressed as both GOx protein content
and nanoreactor GOx activity. The LD\textsubscript{50} values were 0.45
\selectlanguage{greek}µ\selectlanguage{english}g GOx/mL for VLP-GOx and around 0.79 \selectlanguage{greek}µ\selectlanguage{english}g GOx/mL for VLP-GOx@HSA, and no
metabolic activity was detected at 1 \selectlanguage{greek}µ\selectlanguage{english}g GOx/mL for VLP-GOx preparation
(Fig. 6a). The VLP-GOx nanoreactors on 293T kidney cells showed an
LD\textsubscript{50} at around 15 mU of GOx activity per mL of cell
culture, and no metabolic activity at around 30 mU/mL (Fig. 6b). On the
other hand, the VLP-GOx@HSA nanoreactors showed an
LD\textsubscript{50}around 8 mU/mL (Fig. 6b).

\textbf{Hosted file}

\verb`image7.emf`  available at \url{https://authorea.com/users/614980/articles/641602-glucose-oxidase-virus-based-nanoreactors-for-smart-breast-cancer-therapy}

\textbf{Figure 7.} Fluorescence microscopy images of the triple-negative
breast cancer cell line MDA-MB-231 expressing GFP. The images were taken
at different times after treatment with VLP-GOx@HSA. The bubble-like
formations are shown with yellow arrows. See the videos in the
Supplementary information.

The effect of the treatment with VLP-GOx@HSA on a triple-negative breast
cancer cell line (MDA-MB-231) expressing the green fluorescent protein
(GFP) was monitored by fluorescence microscopy (Fig. 7 and videos SI).
After 1-hour treatment in the presence of nanoreactors, the morphology
of cells changed drastically, maybe due to oxidative stress. After 2
hours, bubbles are produced inside the cells suggesting a high
production of hydrogen peroxide, and the fluorescence starts to
diminish. The bubble production is increasing, and the fluorescence
continues to diminish after 3 hours. Finally, after 4 hours the cells
are very damaged, and the fluorescence has completely disappeared.

\textbf{4. Discussion}

Thanks to the complementarity of charges between the negative charge of
the glucose oxidase protein and the positively charged internal side of
the coat protein of BMV, it was possible to encapsulate the GOx inside
the BMV capsid. An average of 0.85 GOx molecules per capsid was
obtained. Considering the hydrodynamic diameter of the dimeric enzyme
([?]12.2 nm) and the internal diameter of the BMV capsid of 17.8
nm,\textsuperscript{{[}45{]}} it seems not possible the encapsulation of
more than one GOx dimeric molecule per capsid. GOx was previously
encapsidated in cowpea chlorotic mottle virus (CCMV) for cascade
reaction study with the so-called DNAzyme, a peroxidase mimic formed in
situ by a specific sequence of ssDNA in the

presence of hemin, and gluconokinase.\textsuperscript{{[}46{]}} However,
no catalytic information of nanoreactors containing only GOx was
reported.

As expected, the nanoreactors showed a higher K\textsubscript{M} than
the free enzyme, especially in the HSA-functionalized nanoreactors. This
could be due to the substrate mass transfer limitations when the enzyme
is inside a protein cage. This mass transfer limitation seems to be also
the origin of the decrease of transformation rate (k\textsubscript{cat})
of nanoreactors VLP-GOx and VLP-GOx@HSA to 14\% and 5.5\% of the value
of free enzyme, respectively. Nevertheless, both preparations showed
high GOx activity.

Unexpectedly, we found a slight cytotoxic effect of unloaded VLPs,
especially on 4T1 cell line (Fig. 4). These results should be taken
cautiously due to the high protein concentration. It is known
that\emph{in vivo} virus and virus-like nanoparticles are able to
enhance specific anti-cancer immune reactions when delivered directly to
the tumor.\textsuperscript{{[}47-49{]}} VLPs from Cowpea mosaic virus
(CPMV) have shown potential as safe anti-cancer immunotherapy (Beatty
and Lewis, 2019).\textsuperscript{{[}50{]}} Systemically administered
CPMV can stimulate an additive therapeutic immune response following
treatment of the tumor with a conventional agent such as
radiation.\textsuperscript{{[}51{]}} However, scarce information is
available on \emph{in vitro} cytotoxicity of empty and unfunctionalized
VPLs. Unfunctionalized P22 VLPs showed a cell survival rate of 90\% of
at concentrations as high as 200 \selectlanguage{greek}μ\selectlanguage{english}g/mL, indicating that VLP had almost
no toxicity to bone marrow-derived dendritic
cells.\textsuperscript{{[}52{]}} On the other hand, unloaded Human
hepatitis B virus-derived VLPs also showed no cytotoxicity on 293 kidney
cell line.\textsuperscript{{[}53{]}}

The LD\textsubscript{50} in both cell lines is similar for VLP-GOx and
VLP-GOx@HSA when evaluated in GOx content or GOx activity (Fig. 4 and
5). Significant damage in cancer cells was found in the triple-negative
breast cancer cell line MDA-MB-231/eGFP (Fig. 6 and videos SI).
MDA-MB-231/eGFP stable cells express a green fluorescent protein (GFP)
as cytoplasmatic proteins. This mammalian cell is transfected with a
cDNA chimera composed of a fusion of the genes encoding the fluorescent
protein and \selectlanguage{greek}β-\selectlanguage{english}actin. After 1-hour incubation in the presence of GOx
enzymatic nanoreactors, a drastic change in the cell morphology is
noted. A plasmatic membrane protrusion after 2 hours of incubation was
clearly observed in phase contrast images (videos SI). This seems to be
due to the formation of gas bubbles inside the cells produced by the
metabolic oxygen production by the antioxidant system (e.g. catalase)
induced by te excess of hydrogen peroxide produced by the GOx activity.
As shown in the videos (SI), when the cell ``explodes'' the fluorescence
immediately disappears due to the cell membrane destruction and the
subsequent dilution of the fluorophore in the medium. Glucose oxidase
has been reported as a cytotoxic agent for tumor cells and its capacity
to reduce the tumor tissue \emph{in
vivo}experiments.\textsuperscript{{[}54{]}} The cytotoxic effect of GOx
is originated from the production of hydrogen peroxide, causing
oxidative damage in the cells, hypoxia as oxygen is consumed, and
glucose consumption that starves the cells.\textsuperscript{{[}55-57{]}}
The advantages of VLPs and nanocarriers of enzymatic activity are the
protection against the proteases,\textsuperscript{{[}38{]}} the
reduction of immunogenic response, especially when the nanoreactors are
covered with compatible proteins or non-immunogenic polymers such as
polyethylene glycol.\textsuperscript{{[}58{]}}

It has been reported that HSA functionalization increases tumor cell
affinity.\textsuperscript{{[}43{]}} The HSA provides an interaction
between the nanocage surface and secreted protein acidic and rich in
cysteine (SPARC). HSA-SPARC interaction allowed breast tumor cell
internalization.\textsuperscript{{[}44{]}} The role of SPARC in cancer
development is still controversial. Higher levels of SPARC expression
have been reported in glioblastomas,\textsuperscript{{[}59{]}}pancreatic
cancer,\textsuperscript{{[}60{]}} gastrointestinal
cancer,\textsuperscript{{[}61{]}} cervical
cancer,\textsuperscript{{[}62{]}} bone
cancer,\textsuperscript{{[}63{]}}and breast
cancer.\textsuperscript{{[}64-66{]}} In contrast, lower levels of SPARC
expression have been found in pancreatic
cancers,\textsuperscript{{[}67{]}} ovarian,\textsuperscript{{[}68{]}}and
colorectal.\textsuperscript{{[}69,70{]}} HSA-based drugs can bind to
SPARC.\textsuperscript{{[}71,72{]}} With this in mind, VLP-GOx
nanoreactors were functionalized with HSA. The HSA covered nanoparticles
(Fig. 3B) significantly increased the protein content of nanoparticles.
However, no advantage in cytotoxicity in cell cultures was found when
the cytotoxicity results were based on GOx content or GOx activity (Fig.
5 and 6). The effect of HSA functionalization \emph{in vivo} experiments
is currently being explored.



\textbf{5. Conclusions}

Self-assembly is a highly efficient procedure for active enzyme
encapsulation. The VLP-encapsulated GOx enzyme showed significant
catalytic activity. The nanoreactors containing GOx activity are
entirely suitable for cytotoxicity generation in tumor cells. The HSA
functionalization of the VLP-GOx nanoreactors could result in a
prevailing strategy to improve selective cancer targeting. The GOx
containing enzymatic nanoreactors seems to be an efficient alternative
to improve the current cancer therapy. \emph{In vivo} studies are
envisaged to reinforce the effectiveness of this treatment strategy.

\textbf{Author contributions}

Pedro Gama: Formal analysis, investigation, methodology, writing --
original draft, writing -- review \& editing. Patricia Juarez: Formal
analysis, investigation, methodology, writing -- review \& editing. Ana
G. Rodr\selectlanguage{ngerman}íguez-Hernández: Investigation, methodology. Rafael
Vazquez-Duhalt: Conceptualization, funding acquisition, investigation,
methodology, project administration, resources, supervision, writing --
review \& editing.

\textbf{Acknowledgments}

This work has been funded by UNAM (grant PAPIIT IN209722). We thank Dr.
Oscar González Davis and Itandehui Betanzo for their technical
assistance, and Francisco Ruiz Medina for TEM analysis.

\textbf{Conflict of interest statement}

The authors declare no competing interests.

\textbf{Data availability statement}

Additional data that support the findings of this study are available
upon reasonable request from the corresponding authors.

\textbf{References}

1. Arnold, M., Morgan, E., Rumgay, H., Mafra, A., Singh, D., Laversanne,
M., Vignat, J., Gralow, J.R., Cardoso, F., Siesling, S. \&
Soerjomataram, I. (2022). Current and future burden of breast cancer:
Global statistics for 2020 and 2040. \emph{The Breast} , 66,15--23.

2. Azamjah, N., Soltan-Zadeh, Y. \& Zayeri, F. (2019). Global trend of
breast cancer mortality rate: A 25-year study. \emph{Asian Pacific
Journal of Cancer Prevention} , 20(7), 2015--2020.

3. Global Cancer Observatory. International Agency for Research on
Cancer. World Health Organization. Data source: 2020.
https://gco.iarc.fr/

4. Corben, A.D. (2013). Pathology of invasive breast
disease.\emph{Surgical Clinics of North America} , 93(2), 363--392.

5. Waks, A.G. \& Winer, E.P. (2019). Breast cancer treatment: A
review.\emph{Journal of the American Medical Association} , 321(3),
288--300.

6. Pearce, A., Haas, M., Viney, R., Pearson, S.A., Haywood, P., Brown,
C. \& Ward, R. (2017). Incidence and severity of self-reported
chemotherapy side effects in routine care: A prospective cohort
study.\emph{PLoS One} , 12(10), e0184360.

7. Altun, \selectlanguage{polish}İ. \selectlanguage{english}\& Sonkaya, A. (2018). The most common side effects
experienced by patients were receiving first cycle of
chemotherapy.\emph{Iranian Journal of Public Health} , 47(8),
1218--1219.

8. Chauhan, K., Sengar, P., Juarez-Moreno K., Hirata, G.A. \&
Vazquez-Duhalt, R. (2020). Camouflaged, activatable and therapeutic
tandem bionanoreactors for breast cancer theranosis. \emph{Journal of
Colloid and Interface Science} , 580, 365--376.

9. Fu, X., Shi, Y., Qi, T., Qiu, S., Huang, Y., Zhao, X., Sun, Q. \&
Lin, G. (2020). Precise design strategies of nanomedicine for improving
cancer therapeutic efficacy using subcellular targeting. \emph{Signal
Transduction and Targeted Therapy} , 5, 262.

10. Tagde, P., Najda, A., Nagpal, K., Kulkarni, G. T., Shah, M., Ullah,
O., Balant, S. \& Rahman, M. H. (2022). Nanomedicine-based delivery
strategies for breast cancer treatment and
management.\emph{International Journal of Molecular Sciences} , 23(5),
2856.

11. Wong, C.M., Wong, K.H. \& Chen, X.D. (2008). Glucose oxidase:
natural occurrence, function, properties and industrial
applications.\emph{Applied Microbiology and Biotechnology} , 78,
927--938.

12. Mano, N. (2019). Engineering glucose oxidase for bioelectrochemical
applications. \emph{Bioelectrochemistry} , 128, 218--240.

13. Fu, L. H., Qi, C., Lin, J. \& Huang, P. (2018). Catalytic chemistry
of glucose oxidase in cancer diagnosis and treatment. \emph{Chemical
Society Reviews} , 47(17), 6454--6472.

14. Dinda, S., Sarkar, S. \& Das, P. K. (2018). Glucose oxidase mediated
targeted cancer-starving therapy by biotinylated self-assembled
vesicles. \emph{Chemical Communications} , 54, 9929--9932.

15. Feng, L., Xie, R., Wang, C., Gai, S., He, F., Yang, D., Yang, P. \&
Lin, J. (2018). Magnetic targeting, tumor microenvironment-responsive
intelligent nanocatalysts for enhanced tumor ablation. \emph{ACS Nano} ,
12 (11), 11000--11012.

16. Chang, K., Liu, Z., Fang, X., Chen, H., Men, X., Yuan, Y., Sun, K.,
Zhang, X., Yuan, Z., \& Wu, C. (2017). Enhanced phototherapy by
nanoparticle-enzyme via generation and photolysis of hydrogen
peroxide.\emph{Nano Letters} 17 (7), 4323--4329.

17. Fu, L.-H., Qi, C., Hu, Y.-R., Lin, J. \& Huang, P. (2019). Glucose
oxidase-instructed multimodal synergistic cancer therapy. \emph{Advanced
Materials} , 31, 1808325.

18. Brigger, I., Dubernet, C. \& Couvreur, P. (2012) Nanoparticles in
cancer therapy and diagnosis. \emph{Advanced Drug Delivery Reviews} ,
64, Suppl. 24--36

19. Ding, X., Liu, D., Booth, G., Gao, W. \& Lu, Y. (2018). Virus-like
particle engineering: from rational design to versatile
applications.\emph{Biotechnology Journal} , 13(5), e1700324.

20. Koellhoffer, E.C. \& Steinmetz, N.F. (2022). Cowpea mosaic virus and
natural killer cell agonism for \emph{in situ} cancer
vaccination.\emph{Nano Letters} , 22(13), 5348--5356.

21. Nkanga, C.I. \& Steinmetz, N.F. (2021). The pharmacology of plant
virus nanoparticles. \emph{Virology} , 556, 39--61.

22. Gonz\selectlanguage{ngerman}ález-Davis, O., Villagrana-Escareño, M.V., Trujillo, M.A., Gama,
P., Chauhan, K. \& Vazquez-Duhalt, R. (2023). Virus-like nanoparticles
as enzyme carriers for Enzyme Replacement Therapy (ERT).\emph{Virology}
, 580, 73--87.

23. Mejía-Méndez, J. L., Vazquez-Duhalt, R., Hernández, L. R.,
Sánchez-Arreola, E. \& Bach, H. (2022). Virus-like particles:
fundamentals and biomedical applications. \emph{International Journal of
Molecular Sciences,} 23(15), 8579.

24. Nooraei, S., Bahrulolum, H., Hoseini1, Z.S., Katalani, C., Hajizade,
A., Easton, A.J. \& Ahmadian, G. (2021). Virus-like particles:
preparation, immunogenicity and their roles as nanovaccines and drug
nanocarriers. \emph{Journal of Nanobiotechnology} , 9, 59.

25. Yu, X., Weng, Z., Zhao, Z., Xu, J., Qi, Z. \& Liu, J. (2022).
Assembly of protein cages for drug delivery. \emph{Pharmaceutics} , 14,
2609.

26. Schoonen, L., \& Van Hest, J.C.M. (2014). Functionalization of
protein-based nanocages for drug delivery applications.\emph{Nanoscale}
, 6(13), 7124--7141.

27. Yan, D., Wei, Y.Q., Guo, H.C. \& Sun, S.Q. (2015). The application
of virus-like particles as vaccines and biological
vehicles.\emph{Applied Microbiology and Biotechnology} , 99(24),
10415--10432.

28. Kraj, P., Selivanovitch, E., Lee, B., \& Douglas, T. (2021). Polymer
coatings on virus-like particle nanoreactors at low ionic
strength---charge reversal and substrate access.\emph{Biomacromolecules}
, 22(5), 2107--2118.

29. Douglas, T. \& Young, M. (2006). Viruses: making friends with old
foes. \emph{Science} , 312(5775), 873--875.

30. Jordan, P., Patterson, D., Saboda, K., Edwards, E., Miettinen, H.,
Basu, G., Thielges, M. \& Douglas, T. (2016). Self-assembling
biomolecular catalysts for hydrogen production. \emph{Nature Chemistry}
, 8, 179--185.

31. Chakraborti, S., Lin, T.-Y., Glatt, S. \& Heddle, J.G. (2020).
Enzyme encapsulation by protein cages. \emph{RSC Advances} , 10(22),
13293--13301.

32. Das, S., Zhao, L., Elofson, K. \& Finn, M.G. (2020). Enzyme
stabilization by virus-like particles. \emph{Biochemistry} , 59 (31),
2870--2881

33. Sánchez-Sánchez, L., Tapia-Moreno, A., Juarez-Moreno, K., Patterson,
D. P., Cadena-Nava, R. D., Douglas, T. \& Vazquez-Duhalt, R. (2015).
Design of a VLP-nanovehicle for CYP450 enzymatic activity
delivery.\emph{Journal of Nanobiotechnology} , 13(1), 1--10.

34. Tapia-Moreno, A., Juarez-Moreno, K., Gonzalez-Davis, O.,
Cadena-Nava, R. D. \& Vazquez-Duhalt, R. (2017). Biocatalytic virus
capsid as nanovehicle for enzymatic activation of Tamoxifen in tumor
cells. \emph{Biotechnology Journal} , 12, 1600706.

35. Chauhan, K., Hernandez-Meza, J.M., Rodríguez-Hernández, A.G.,
Juarez\selectlanguage{english}-Moreno, K., Sengar, P. \& Vazquez\selectlanguage{english}-Duhalt, R. (2018).
Multifunctionalized biocatalytic P22 nanoreactor for combinatory
treatment of ER+ breast cancer. \emph{Journal of Nanobiotechnology} ,
16, 17.

36. Schwarz, B., Uchida, M. \& Douglas T. (2017). Biomedical and
catalytic opportunities of virus-like particles in
nanotechnology.\emph{Advances in Virus Research} , 97, 1--60.

37. Inoue, T., Kawano, M.A., Takahashi, R.U., Tsukamoto, H., Enomoto,
T., Imai, T., Kataoka, K. \& Handa, H. (2008). Engineering of SV40-based
nano-capsules for delivery of heterologous proteins as fusions with the
minor capsid proteins VP2/3. \emph{Journal of Biotechnology} , 134(1-2),
181--92.

38. O'Neil, A., Prevelige, P. E. \& Douglas, T. (2013). Stabilizing
viral nano-reactors for nerve-agent degradation. \emph{Biomaterials
Science} , 1(8), 881--886.

39. Selivanovitch, E., LaFrance, B. \& Douglas, T. (2021). Molecular
exclusion limits for diffusion across a porous capsid. \emph{Nature
Communications} , 12, 2903.

40. Gama, P., Cadena-Nava, R. D., Juarez-Moreno, K., Perez-Robles, J. \&
Vazquez-Duhalt, R. (2021). Virus-based nanoreactors with GALT activity
for classic galactosemia therapy. \emph{ChemMedChem} , 16, 1438.

41. Vervoort, D.F.M., Heiringhoff, R., Timmermans, S., van Stevendaal,
M. \& van Hest J.C.M. (2021). Dual site-selective presentation of
functional handles on protein-engineered Cowpea chlorotic mottle
virus-like particles. \emph{Bioconjugate Chemistry} , 32 (5), 958--963.

42. Cadena-Nava, R. D., Comas-Garcia, M., Garmann, R. F., Rao, A. L. N.,
Knobler, C. M. \& Gelbart, W. M. (2012). Self-Assembly of Viral Capsid
Protein and RNA Molecules of Different Sizes: Requirement for a Specific
High Protein/RNA Mass Ratio. \emph{Journal of Virology} , 86(6),
3318--3326.

43. Zhou, C., Song, X., Guo, C., Tan, Y., Zhao, J., Yang, Q., Chen, D.,
Tan, T., Sun, X., Gong, T. \& Zhang, Z. (2019). An alternative and
injectable preformed albumin-bound anticancer drug delivery system for
anticancer and antimetastasis treatment. \emph{ACS Applied Materials \&
Interfaces,} 11 (45), 42534--42548.

44. Spada, A., Emami, J., Tuszynski, J. A. \& Lavasanifar, A. (2021).
The uniqueness of albumin as a carrier in nanodrug
delivery.\emph{Molecular Pharmaceutics} , 8 (5), 1862--1894.

45. Lucas, R. W., Larson, S. B. \& McPherson, A. (2002). The
crystallographic structure of brome mosaic virus. \emph{Journal of
Molecular Biology} , 317(1), 95--108.

46. Brasch, M., Putri, R.M., de Ruiter, M.V., Luque, D., Koay, M.S.T.,
Caston, J.R., \& Cornelissen, J.J.L.M. (2017) Assembling enzymatic
cascade pathways inside virus-based nanocages using dual-tasking nucleic
acid tags. \emph{Journal of the American Chemistry Society} ,
139(4),1512-1519.

47. Al-Barwani, F., Donaldson, B., Pelham, S.J., Young, S.L. \& Ward,
V.K. (2014) Antigen delivery by virus-like particles for
immunotherapeutic vaccination. \emph{Therapy Delivery} 5(11),
1223--1240.

48. Mohsen, M.O., Speiser, D.E., Knuth, A. \& Bachmann, M.F. (2020)
Virus-like particles for vaccination against cancer. \emph{Wiley
Interdisciplinary Review of Nanomedicine and Nanobiotechnology} 12,
1579.

49. Duval, K.E.A., Wagner, R.J., Beiss, V., Fiering, S.N., Steinmetz,
N.F. \& Hoopes, P.J. (2020) Cowpea mosaic virus nanoparticle enhancement
of hypofractionated radiation in a B16 murine melanoma
model.\emph{Frontiers in Oncology.} 16(10), 594614.

50. Beatty, P.H. \& Lewis, J.D. (2019) Cowpea mosaic virus nanoparticles
for cancer imaging and therapy. \emph{Advances in Drug Delivery
Review}145, 130--144.

51. Patel, R., Czapar, A.E., Fiering, S., Oleinick, N.L. \& Steinmetz,
N.F. (2018) Radiation therapy combined with cowpea mosaic virus
nanoparticle in situ vaccination initiates immune-mediated tumor
regression. \emph{ACS Omega} 3(4), 3702-3707.

52. Li, W., Jing, Z., Wang, S., Li, Q., Xing, Y., Shi, H., Li, S. \&
Hong, Z. (2021) P22 virus-like particles as an effective antigen
delivery nanoplatform for cancer immunotherapy. \emph{Biomaterials} 271,
120726.

53. Sakai, C., Hosokawa, K., Watanabe, T., Suzuki, Y., Nakano, T., Ueda,
K. \& Fujimuro, M. (2021) Human hepatitis B virus-derived virus-like
particle as a drug and DNA delivery carrier. \emph{Biochemical and
Biophysical Research Communications.} 581, 103--109.

54. Huo, M., Wang, L., Chen, Y. \& Shi, J. (2017). Tumor-selective
catalytic nanomedicine by nanocatalyst delivery. \emph{Nature
Communications} , 8(1), 357.

55. Zhao, W., Hu, J. \& Gao, W. (2017). Glucose oxidase-polymer nanogels
for synergistic cancer-starving and oxidation therapy. \emph{ACS Applied
Materials \& Interfaces} , (28), 23528--23535.

56. Zhang, R., Feng, L., Dong, Z., Wang, L., Liang, C., Chen, J., Ma,
Q., Zhang, R., Chen, Q., Wang, Y. \& Liu, Z. (2018). Glucose \& oxygen
exhausting liposomes for combined cancer starvation and
hypoxia-activated therapy. \emph{Biomaterials} , 162, 123--131.

57. Fan, W., Lu, N., Huang, P., Liu, Y., Yang, Z., Wang, S., Yu, G.,
Liu, Y., Hu, J., He, Q., Qu, J., Wang, T. \& Chen, X. (2017).
Glucose-responsive sequential generation of hydrogen peroxide and Nitric
Oxide for Synergistic Cancer Starving-Like/Gas Therapy. \emph{Angewandte
Chemie International Edition} , 56(5), 1229--1233.

58. Nunez-Rivera, A., Fournier, P. G. J., Arellano, D. L.,
Rodriguez-Hernandez, A. G., Vazquez-Duhalt, R. \& Cadena-Nava, R. D.
(2020). Brome mosaic virus-like particles as siRNA nanocarriers for
biomedical purposes. \emph{Beilstein Journal of Nanotechnology} , 11,
372--382.

59. Rempel, S. A., Golembieski, W. A., Ge, S., Lemke, N., Elisevich, K.,
Mikkelsen, T. \& Gutierrez, J. A. (1998). SPARC: A Signal of Astrocytic
Neoplastic Transformation and Reactive Response in Human Primary and
Xenograft Gliomas. \emph{Journal of Neuropathology and Experimental
Neurology} , 57(12), 1112--1121.

60. Von Hoff, D. D., Ramanathan, R. K., Borad, M. J., Laheru, D. A.,
Smith, L. S., Wood, T. E., Korn, R. L., Desai, N., Trieu, V., Iglesias,
J. L., Zhang, H., Soon-Shiong, P., Shi, T., Rajeshkumar, N. V., Maitra,
A. \& Hidalgo, M. (2011). Gemcitabine Plus nab-Paclitaxel Is an Active
Regimen in Patients With Advanced Pancreatic Cancer: A Phase I/II
Trial.\emph{Journal of Clinical Oncology} , 29(34), 4548--4554.

61. Gamradt, P., De La Fouchardiere, C. \& Hennino, A. (2021). Stromal
Protein-Mediated Immune Regulation in Digestive Cancers. \emph{Cancers}
, 13 (1), 146.

62. Sova, P., Feng, Q., Geiss, G., Wood, T., Strauss, R., Rudolf, V.,
Lieber, A., \& Kiviat, N. (2006). Discovery of Novel Methylation
Biomarkers in Cervical Carcinoma by Global Demethylation and Microarray
Analysis. \emph{Cancer Epidemiology, Biomarkers \& Prevention} , 15 (1),
114--123.

63. Bellahcene, A. \& Castronovo V. (1995). Increased expression of
osteonectin and osteopontin, two bone matrix proteins, in human breast
cancer. \emph{The American Journal of Pathology} , 146(1), 95--100.

64. Lien, H. C., Hsiao, Y. H., Lin, Y. S., Yao, Y.T., Juan, H. F., Kuo,
W. H., Hung, M., Chang, K. J. \& Hsieh, F. J. (2007). Molecular
Signatures of Metaplastic Carcinoma of the Breast by Large-Scale
Transcriptional Profiling: Identification of Genes Potentially Related
to Epithelial-Mesenchymal Transition. \emph{Oncogene} , 26, 7859--7871.

65. Jones, C., Mackay, A., Grigoriadis, A., Cossu, A., Reis-Filho, J.
S., Fulford, L., Dexter, T., Davies, S., Bulmer, K., Ford, E., Parry,
S., Budroni, M., Palmieri, G., Neville, A. M., O'Hare, M. J. \& Lakhani,
S. R. (2004). Expression Profiling of Purified Normal Human Luminal and
Myoepithelial Breast Cells Identification of Novel Prognostic Markers
for Breast Cancer. \emph{Cancer Research} , 64 (9), 3037--3045.

66. Shi, S., Ma, H.-Y., Han, X.-Y., Sang, Y.-Z., Yang, M.-Y. \& Zhang,
Z.-G. (2022). Prognostic Significance of SPARC Expression in Breast
Cancer: A Meta-Analysis and Bioinformatics Analysis. \emph{BioMed
Research International} , 2022, 8600419.

67. Puolakkainen, P. A., Brekken, R. A., Muneer, S., \& Sage, E. H.
(2004). Enhanced Growth of Pancreatic Tumors in SPARC-Null Mice Is
Associated with Decreased Deposition of Extracellular Matrix and Reduced
Tumor Cell Apoptosis. \emph{Molecular Cancer Research} , 2, 215--224.

68. Yiu, G. K., Chan, W. Y., Ng, S. W., Chan, P. S., Cheung, K. K.,
Berkowitz, R. S. \& Mok S. C. (2001). SPARC (secreted protein acidic and
rich in cysteine) induces apoptosis in ovarian cancer cells. \emph{The
American Journal of Pathology} , 159(2), 609--622.

69. Yang, E., Kang, H. J., Koh, K. H., Rhee, H., Kim, N. K. \& Kim, H.
(2007). Frequent inactivation of SPARC by promoter hypermethylation in
colon cancers. \emph{The International Journal of Cancer} , 121,
567--575.

70. Cheetham, S., Tang, M. J., Mesak, F., Kennecke, H., Owen, D., \&
Tai, I. T. (2008). SPARC promoter hypermethylation in colorectal cancers
can be reversed by 5-Aza-2\selectlanguage{english}'deoxycytidine to increase SPARC expression
and improve therapy response. \emph{British Journal of Cancer} , 98,
1810--1819.

71. Tao, H., Wang, R., Sheng, W., \& Zhen, Y. (2021). The development of
human serum albumin-based drugs and relevant fusion proteins for cancer
therapy. \emph{International Journal of Biological Macromolecules} ,
187, 24--34.

72. Are, R. P. \& Babu, A. R. (2022). Molecular Interaction Analysis of
SPARC--Collagen with Human Serum Albumin. \emph{Journal of Computational
Biophysics and Chemistry} , 21 (8), 927--939.

\textbf{Figure leyends}

\textbf{Figure 1.} a) Surface electric charge distribution on the GOx
enzyme and BMV capsomer. The GOx surface mostly exhibits negative
electric charges (red color), and the inner surface of BMV capsomers
shows a more positive (blue color) electric charge distribution. The
difference in electric charge distribution between GOx and the inner BMV
surface results in electric charge complementarity. In silico results by
Maestro Schrodinger(r). b) Schematic representation of VLP-GOx nanoreactor
self-assembly encapsulation and HSA functionalization

\textbf{Figure 2.} Purification of VLP-GOx nanoreactors by SEC HPLC
chromatography using differences in retention time A) Overlapped
chromatograms show the retention time for native BMV=5.7 min, free
GOx=7.5 min and disassembled CP=8.4 min. B) VLP-GOx nanoreactors
retention time=5.7 min, similar to native virions of BMV. The VLP-GOx
nanoreactors were purified by collecting the fractions from 5.5 to 6.5
min.

\textbf{Figure 3.} Transmission electron microscopy (TEM) of
nanoreactors. A) Quasi-spherical unmodified VLP-GOx nanoreactors, and B)
VLP-GOx@HSA functionalized nanoreactors.

\textbf{Figure 4.} Cytotoxicity activity of empty unmodified and
modified VLPs on cell cultures of 4T1 breast tumor mouse cell line
model, and 293T healthy embryonic kidney human cells. Two-way RM ANOVA
(p\textless{}0.001). *** Significative difference between the two
preparations at the same concentration. \selectlanguage{english}++ Significative difference with
the control.

\textbf{Figure 5.} Cytotoxicity of unfuctionalized VLP-GOx and
functionalized VLP-GOx@HSA nanoreactors on 4T1 breast tumor mouse cell
line. Mean and standard deviation were obtained from three independent
experiments.

\textbf{Figure 6.} Cytotoxicity of unfuctionalized VLP-GOx and
functionalized VLP-GOx@HSA nanoreactors on 293T kidney cell line. Mean
and standard deviation were obtained from three independent experiments.

\textbf{Figure 7.} Flourescence microscopy images of the triple negative
breast cancer cell line MDA-MB-231 expressing GFP. The images were taken
at different times after treatement with VLP-GOx@HSA. The bubble-like
formations are shown with yellow arrows. See videos on the Supplementary
information.

\selectlanguage{english}
\FloatBarrier
\end{document}

